Sinoatrial node (SAN) cells are cardiac pacemaker cells located in the lateral right atrium that require proper Ca2+ handling to produce a rhythmic heart rate (HR). It is well accepted that SAN cells rely on slow depolarizing If channels, sarcoplasmic Ca2+ release and modulation of intracellular Ca2+ by membrane ion channels to modulate HR. The mitochondrial calcium uniporter (MCU) is a newly identified 40 kDa protein that is responsible for Ca2+ influx into mitochondria. I found that inhibiting MCU with the antagonist drug, RU360, blocks isolated SAN cells from increasing beating frequency in response to isoproterenol, a -adrenergic receptor agonist. This outcome was reversed when ATP was perfused in the pipette solution. Based on these data, I hypothesize that mitochondrial Ca2+ is a required signal for SAN cells to respond to isoproterenol and that inhibiting MCU prevents mitochondria from matching energy supply with demand. Sinus node dysfunction (SND) is a clinical condition marked by dysregulation of the primary cardiac pacemaker, slowing of the HR and increased risk of arrhythmias and sudden death. At present the only treatment for SND is surgical implantation of a permanent pacemaker, which costs 2 billion dollars annually and is associated with numerous morbidities. Our lab recently reported a novel mechanism in which angiotensin II (Ang II) infused mice developed SND due to SAN cell death. Mitochondrial Ca2+ is a required signal for regulation of metabolic processes, but excessive mitochondrial Ca2+ load can lead to mitochondrial induced cell death. Here I will test the novel concept that the MCU is a gatekeeper for mitochondrial Ca2+ entry but with the potential to permit mitochondrial Ca2+ overload, cell death and SND. I developed mice with genetic MCU inhibition, by myocardial-delimited transgenic expression of a dominant-negative MCU. I will use these new mice to test the potential role of MCU in SAN physiology and in Ang-II induced SND. To my knowledge, this will be the first in vivo study of the MCU in cardiac tissue. The goal of thi work is to determine how mitochondrial Ca2+ contributes to cardiac pacing and explore the effect of MCU inhibition in an Ang II infusion model of SND. My studies will test the hypothesized role of the MCU in SAN physiology and disease using two Specific Aims: Aim 1: Determine the physiologic effects of inhibiting Ca2+ influx through the MCU. Aim 2: Determine whether MCU inhibition affects physiological HR responses and/or protects against SAN cell death and SND in vivo.